Sarcomeric Auto-Oscillations in Single Myofibrils From the Heart of Patients With Dilated Cardiomyopathy.

BACKGROUND: Left ventricular wall motion is depressed in patients with dilated cardiomyopathy (DCM). However, whether or not the depressed left ventricular wall motion is caused by impairment of sarcomere dynamics remains to be fully clarified. METHODS AND RESULTS: We analyzed the mechanical properties of single sarcomere dynamics during sarcomeric auto-oscillations (calcium spontaneous oscillatory contractions [Ca-SPOC]) that occurred at partial activation under the isometric condition in myofibrils from donor hearts and from patients with severe DCM (New York Heart Association classification III-IV). Ca-SPOC reproducibly occurred in the presence of 1 µmol/L free Ca2+ in both nonfailing and DCM myofibrils, and sarcomeres exhibited a saw-tooth waveform along single myofibrils composed of quick lengthening and slow shortening. The period of Ca-SPOC was longer in DCM myofibrils than in nonfailing myofibrils, in association with prolonged shortening time. Lengthening time was similar in both groups. Then, we performed Tn (troponin) exchange in myofibrils with a DCM-causing homozygous mutation (K36Q) in cTnI (cardiac TnI). On exchange with the Tn complex from healthy porcine ventricles, period, shortening time, and shortening velocity in cTnI-K36Q myofibrils became similar to those in Tn-reconstituted nonfailing myofibrils. Protein kinase A abbreviated period in both Tn-reconstituted nonfailing and cTnI-K36Q myofibrils, demonstrating acceleration of cross-bridge kinetics. CONCLUSIONS: Sarcomere dynamics was found to be depressed under loaded conditions in DCM myofibrils because of impairment of thick-thin filament sliding. Thus, microscopic analysis of Ca-SPOC in human cardiac myofibrils is beneficial to systematically unveil the kinetic properties of single sarcomeres in various types of heart disease.

Spontaneous oscillatory contraction (SPOC) in cardiomyocytes.

SPOC (spontaneous oscillatory contraction) is a characteristic state of the contractile system of striated (skeletal and cardiac) muscle that exists between the states of relaxation and contraction. For example, Ca-SPOCs occur at physiological Ca2+ levels (pCa ∼6.0), whereas ADP-SPOC occurs in the virtual absence of Ca2+ (pCa ≥ 8; relaxing conditions in the presence of MgATP), but in the presence of inorganic phosphate (Pi) and a high concentration of MgADP. The concentration of Mg-ADP necessary for SPOC is nearly equal to or greater than the MgATP concentration for cardiac muscle and is several times higher for skeletal muscle. Thus, the cellular conditions for SPOC are broader in cardiac muscle than in skeletal muscle. During these SPOCs, each sarcomere in a myofibril undergoes length oscillation that has a saw-tooth waveform consisting of a rapid lengthening and a slow shortening phase. The lengthening phase of one half of a sarcomere is transmitted to the adjacent half of the sarcomere successively, forming a propagating wave (termed a SPOC wave). The SPOC waves are synchronized across the cardiomyocytes resulting in a visible wave of successive contractions and relaxations termed the SPOC wave. Experimentally, the SPOC period (and therefore the velocity of SPOC wave) is observed in demembranated cardiomyocytes and can be prepared from a wide range of animal hearts. These periods correlate well with the resting heartbeats of a wide range of mammals (rat, rabbit, dog, pig and cow). Preliminary experiments showed that the SPOC properties of human cardiomyocytes are similar to the heartbeat of a large dog or a pig. This correlation suggests that SPOCs may play a fundamental role in the heart. Here, we briefly summarize a range of SPOC parameters obtained experimentally, and relate them to a theoretical model to explain those characteristics. Finally, we discuss the possible significance of these SPOC properties in each and every heartbeat.

Depressed Frank-Starling mechanism in the left ventricular muscle of the knock-in mouse model of dilated cardiomyopathy with troponin T deletion mutation ΔK210.

It has been reported that the Frank-Starling mechanism is coordinately regulated in cardiac muscle via thin filament "on-off" equilibrium and titin-based lattice spacing changes. In the present study, we tested the hypothesis that the deletion mutation ΔK210 in the cardiac troponin T gene shifts the equilibrium toward the "off" state and accordingly attenuate the sarcomere length (SL) dependence of active force production, via reduced cross-bridge formation. Confocal imaging in isolated hearts revealed that the cardiomyocytes were enlarged, especially in the longitudinal direction, in ΔK210 hearts, with striation patterns similar to those in wild type (WT) hearts, suggesting that the number of sarcomeres is increased in cardiomyocytes but the sarcomere length remains unaltered. For analysis of the SL dependence of active force, skinned muscle preparations were obtained from the left ventricle of WT and knock-in (ΔK210) mice. An increase in SL from 1.90 to 2.20µm shifted the mid-point (pCa50) of the force-pCa curve leftward by ~0.21pCa units in WT preparations. In ΔK210 muscles, Ca(2+) sensitivity was lower by ~0.37pCa units, and the SL-dependent shift of pCa50, i.e., ΔpCa50, was less pronounced (~0.11pCa units), with and without protein kinase A treatment. The rate of active force redevelopment was lower in ΔK210 preparations than in WT preparations, showing blunted thin filament cooperative activation. An increase in thin filament cooperative activation upon an increase in the fraction of strongly bound cross-bridges by MgADP increased ΔpCa50 to ~0.21pCa units. The depressed Frank-Starling mechanism in ΔK210 hearts is the result of a reduction in thin filament cooperative activation.

Sarcomere imaging by quantum dots for the study of cardiac muscle physiology.

We here review the use of quantum dots (QDs) for the imaging of sarcomeric movements in cardiac muscle. QDs are fluorescence substances (CdSe) that absorb photons and reemit photons at a different wavelength (depending on the size of the particle); they are efficient in generating long-lasting, narrow symmetric emission profiles, and hence useful in various types of imaging studies. Recently, we developed a novel system in which the length of a particular, single sarcomere in cardiomyocytes can be measured at ~30 nm precision. Moreover, our system enables accurate measurement of sarcomere length in the isolated heart. We propose that QDs are the ideal tool for the study of sarcomere dynamics during excitation-contraction coupling in healthy and diseased cardiac muscle.

Real-time measurement of the length of a single sarcomere in rat ventricular myocytes: a novel analysis with quantum dots.

As the dynamic properties of cardiac sarcomeres are markedly changed in response to a length change of even ∼0.1 µm, it is imperative to quantitatively measure sarcomere length (SL). Here we show a novel system using quantum dots (QDs) that enables a real-time measurement of the length of a single sarcomere in cardiomyocytes. First, QDs were conjugated with anti-α-actinin antibody and applied to the sarcomeric Z disks in isolated skinned cardiomyocytes of the rat. At partial activation, spontaneous sarcomeric oscillations (SPOC) occurred, and QDs provided a quantitative measurement of the length of a single sarcomere over the broad range (i.e., from ∼1.7 to ∼2.3 µm). It was found that the SPOC amplitude was inversely related to SL, but the period showed no correlation with SL. We then treated intact cardiomyocytes with the mixture of the antibody-QDs and FuGENE HD, and visualized the movement of the Z lines/T tubules. At a low frequency of 1 Hz, the cycle of the motion of a single sarcomere consisted of fast shortening followed by slow relengthening. However, an increase in stimulation frequency to 3-5 Hz caused a phase shift of shortening and relengthening due to acceleration of relengthening, and the waveform became similar to that observed during SPOC. Finally, the anti-α-actinin antibody-QDs were transfected from the surface of the beating heart in vivo. The striated patterns with ∼1.96-µm intervals were observed after perfusion under fluorescence microscopy, and an electron microscopic observation confirmed the presence of QDs in and around the T tubules and Z disks, but primarily in the T tubules, within the first layer of cardiomyocytes of the left ventricular wall. Therefore, QDs are a useful tool to quantitatively analyze the movement of single sarcomeres in cardiomyocytes, under various experimental settings.